PROJECT SUMMARY Pathogenic bacteria employ myriad processes to cause human disease, and these processes include biofilm formation, chemotaxis and motility, and the implementation of secretion systems, all of which ultimately aid in establishing intimate associations between the bacterium and the host. Several medically important bacteria, including some human pathogenic Alphaproteobateria, are extremely difficult to grow, handle, and/or manipulate, and thus, many of the basic virulence mechanisms related to the processes mentioned above are poorly defined in these organisms. Therefore, there are many benefits, including novel therapeutic and vaccine development that may be gleaned from defining broadly conserved bacterial virulence mechanisms. Considering members of the Alphaproteobacteria, the plant pathogen Agrobacterium tumefaciens serves as an exemplary model for studying basic virulence mechanisms that are highly conserved among the Alphaproteobacteria, including those causing human disease. A. tumefaciens is a free-living soil bacterium that senses and responds to signals released from wounded plants, resulting in bacterial chemotaxis toward the wounded plant. A. tumefaciens subsequently employs a type IV secretion system to transfer a T- DNA complex into the cells of the plant. This conjugation event leads to the formation of crown gall tumors, which are severely detrimental to the plant. Preliminary results indicate a LysR-type transcriptional regulator, VtlR (for virulence-associated transcriptional LysR-family regulator) is required for efficient A. tumefaciens-mediated tumor formation. Preliminary data demonstrate that an A. tumefaciens vtlR deletion strain produces fewer and less robust tumors in potatoes compared to the parental strain, and moreover, the vtlR deletion strain exhibits significant defects in biofilm formation. Transcriptomic analyses revealed that VtlR controls the expression of a small regulatory RNA (sRNA) called AbcR1, as well as a multitude of other genes related to conjugation systems, nutrient uptake, and virulence. The VtlR orthologue in the closely related alphaproteobacterium Brucella abortus is required for virulence in an animal model of infection, and thus, VtlR-family regulators are highly conserved and appear to be required for host-bacteria interactions in the Alphaproteobacteria. Therefore, the systematic characterization of VtlR in A. tumefaciens will significantly illuminate the mechanism by which this transcriptional regulator is linked to virulence in Alphaproteobacteria.